TY - JOUR
T1 - Pursuing Advances in DNA Sequencing Technology to Solve a Complex Genomic Jigsaw Puzzle
T2 - The Agglutinin-Like Sequence (ALS) Genes of Candida tropicalis
AU - Oh, Soon Hwan
AU - Isenhower, Allyson
AU - Rodriguez-Bobadilla, Rubi
AU - Smith, Brooke
AU - Jones, Jillian
AU - Hubka, Vit
AU - Fields, Christopher
AU - Hernandez, Alvaro
AU - Hoyer, Lois L.
N1 - Funding Information:
AI, RR-B, BS, and JJ were part of the Undergraduate Program in Fungal Genomics, which is a collaboration between the University of Illinois at Urbana-Champaign and Millikin University. Other students who contributed to this effort included Kaia Ball, Madeline Batek, Anton Bershanskiy, Stephen DeMartini, Erica Forbes, Zeidy Garcia, Jessie Kirk, Mariah McNamer, Deniz Namik, and Quinn Nguyen. The authors thank Drs. Travis Wilcoxen and Laura Zimmerman, Millikin University Department of Biology, for coordinating student participation and supervising research credit for students in the program. The authors thank Pat Kammeyer (Loyola University Medical Center), David Coleman (Trinity College Dublin), and David Soll (University of Iowa) for providing the fungal isolates. Funding. This work was funded by R15 DE026401 from the National Institute of Dental and Craniofacial Research, National Institutes of Health.
Publisher Copyright:
© Copyright © 2021 Oh, Isenhower, Rodriguez-Bobadilla, Smith, Jones, Hubka, Fields, Hernandez and Hoyer.
PY - 2021/1/20
Y1 - 2021/1/20
N2 - The agglutinin-like sequence (ALS) gene family encodes cell-surface adhesins that interact with host and abiotic surfaces, promoting colonization by opportunistic fungal pathogens such as Candida tropicalis. Studies of Als protein contribution to C. tropicalis adhesion would benefit from an accurate catalog of ALS gene sequences as well as insight into relative gene expression levels. Even in the genomics era, this information has been elusive: genome assemblies are often broken within ALS genes because of their extensive regions of highly conserved, repeated DNA sequences and because there are many similar ALS genes at different chromosomal locations. Here, we describe the benefit of long-read DNA sequencing technology to facilitate characterization of C. tropicalis ALS loci. Thirteen ALS loci in C. tropicalis strain MYA-3404 were deduced from a genome assembly constructed from Illumina MiSeq and Oxford Nanopore MinION data. Although the MinION data were valuable, PCR amplification and Sanger sequencing of ALS loci were still required to complete and verify the gene sequences. Each predicted Als protein featured an N-terminal binding domain, a central domain of tandemly repeated sequences, and a C-terminal domain rich in Ser and Thr. The presence of a secretory signal peptide and consensus sequence for addition of a glycosylphosphatidylinositol (GPI) anchor was consistent with predicted protein localization to the cell surface. TaqMan assays were designed to recognize each ALS gene, as well as both alleles at the divergent CtrALS3882 locus. C. tropicalis cells grown in five different in vitro conditions showed differential expression of various ALS genes. To place the C. tropicalis data into a larger context, TaqMan assays were also designed and validated for analysis of ALS gene expression in Candida albicans and Candida dubliniensis. These comparisons identified the subset of highly expressed C. tropicalis ALS genes that were predicted to encode proteins with the most abundant cell-surface presence, prioritizing them for subsequent functional analysis. Data presented here provide a solid foundation for future experimentation to deduce ALS family contributions to C. tropicalis adhesion and pathogenesis.
AB - The agglutinin-like sequence (ALS) gene family encodes cell-surface adhesins that interact with host and abiotic surfaces, promoting colonization by opportunistic fungal pathogens such as Candida tropicalis. Studies of Als protein contribution to C. tropicalis adhesion would benefit from an accurate catalog of ALS gene sequences as well as insight into relative gene expression levels. Even in the genomics era, this information has been elusive: genome assemblies are often broken within ALS genes because of their extensive regions of highly conserved, repeated DNA sequences and because there are many similar ALS genes at different chromosomal locations. Here, we describe the benefit of long-read DNA sequencing technology to facilitate characterization of C. tropicalis ALS loci. Thirteen ALS loci in C. tropicalis strain MYA-3404 were deduced from a genome assembly constructed from Illumina MiSeq and Oxford Nanopore MinION data. Although the MinION data were valuable, PCR amplification and Sanger sequencing of ALS loci were still required to complete and verify the gene sequences. Each predicted Als protein featured an N-terminal binding domain, a central domain of tandemly repeated sequences, and a C-terminal domain rich in Ser and Thr. The presence of a secretory signal peptide and consensus sequence for addition of a glycosylphosphatidylinositol (GPI) anchor was consistent with predicted protein localization to the cell surface. TaqMan assays were designed to recognize each ALS gene, as well as both alleles at the divergent CtrALS3882 locus. C. tropicalis cells grown in five different in vitro conditions showed differential expression of various ALS genes. To place the C. tropicalis data into a larger context, TaqMan assays were also designed and validated for analysis of ALS gene expression in Candida albicans and Candida dubliniensis. These comparisons identified the subset of highly expressed C. tropicalis ALS genes that were predicted to encode proteins with the most abundant cell-surface presence, prioritizing them for subsequent functional analysis. Data presented here provide a solid foundation for future experimentation to deduce ALS family contributions to C. tropicalis adhesion and pathogenesis.
KW - ALS genes
KW - Candida tropicalis
KW - fungal adhesion
KW - gene expression
KW - genome
UR - http://www.scopus.com/inward/record.url?scp=85100595259&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85100595259&partnerID=8YFLogxK
U2 - 10.3389/fmicb.2020.594531
DO - 10.3389/fmicb.2020.594531
M3 - Article
C2 - 33552012
AN - SCOPUS:85100595259
VL - 11
JO - Frontiers in Microbiology
JF - Frontiers in Microbiology
SN - 1664-302X
M1 - 594531
ER -